CN105733987A - Mycoplasma synoviae - Google Patents
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Abstract
The invention provides mycoplasma synoviae.A preservation number of the mycoplasma synoviae is CCTCC M 2016080.The mycoplasma synoviae has the advantages that a mycoplasma synoviae YBF-MS1 strain is stable in biological characteristics, high in toxicity and excellent in immunogenicity; oil adjuvant inactivated vaccine prepared from the mycoplasma synoviae is safe and reliable, is long in persistent period and has cross protection properties, high-level antibodies can be generated by the oil adjuvant inactivated vaccine, the post-immunization mycoplasma synoviae morbidity can be obviously reduced, and accordingly epidemic mycoplasma synoviae can be effectively prevented; mycoplasma synoviae vaccine is not available on market in China, the mycoplasma synoviae inactivated vaccine prepared from the mycoplasma synoviae YBF-MS1 strain is a key for preventing and controlling diseases, and market gaps can be filled.
Description
Technical field
The invention belongs to technical field of vaccines, be specifically related to a kind of chicken Mycoplasma synoviae.
Background technology
Chicken Mycoplasma synoviae (Mycoplasma synoviae, MS), also known as avian infectious synovitis, is the acute or chronic infectious disease of one being caused chicken and turkey etc. by chicken Mycoplasma synoviae.Infectiousness synovitis is that (1964) such as Olson are first reported.Primary disease is to encroach on knuckle synovia, and stndon sheath film is main, it is characterized by joint, stndon sheath and vola swelling etc..Chicken group infects this disease and may result in significantly limping, growth retardation and trunk degradation etc..Chicken Mycoplasma synoviae infects and extends over the entire globe, and in worldwide distribution, mainly encroaches on commercial meat bird, laying hen and kind chicken, and primary disease the most all can occur, but easily send out with winter-spring season.Chicken Mycoplasma synoviae infects the chicken being typically more common in 4～16 week old, and sickness rate is generally 5%～15%, and mortality rate is about 1%～10%.Adult Chicken mostly is chronic or inapparent infection.Due to primary disease slower development, the course of disease is long, once infects in chicken group, eradicates highly difficult, therefore spread for a long time in chicken group, causes that efficiency of feed utilization is low, growth retardation, mortality increase, degradation under egg production.When chicken group such as exists this cause of disease, mixed infection easily occurring, aggravates one's illness, mortality rate increases, and causes serious economic loss.Chicken Mycoplasma synoviae cause of disease is Mycoplasma synoviae, and this bacterium is polymorphic spherical bacterium through the thalli morphology that Ji's nurse Sa dyes, diameter about 0.2 μm.The pathogenicity of different strains difference, the symptom caused is also different because of the tropism of cause of disease.I.e. glucose fermentation, does not hydrolyze arginine, does not utilize carbamide.During artificial culture, the nutritional requirement of MS is higher.Must be added to nicotinamide adenine dinucleotide (nadide) in culture medium, the most also need to add horse or the serum of pig, the serum with pig is the best, and the suitableeest cultivation temperature is 37 DEG C.
Document announcement chicken synovial fluid mycoplasma is S type in the seroclassification of avian mycoplasmas, and Mycoplasma synoviae only has 1 serotype, shows through DNA-DNA cross experiment, almost without difference between different bacterial strains.Although chicken Mycoplasma synoviae is at present at China's also not epidemic data the most widely, but existing from Henan, Shaanxi, Beijing, Guangdong, Guangxi, the area such as Huhehaote and Shanghai be separated to the report of MS.Ning Yibao etc. (1986), through serology, the survey showed that, and China's Mycoplasma synoviae the rate of positive infection is 20.7%, generally exists in a lot of area chicken groups at home, and some areas are the most serious.Chen Jianhong etc. (2001), the 185 example blood serum samples to Delta of the Pearl River some areas gaggle, chicken Mycoplasma synoviae is infected and has carried out Serologic detection by application plate agglutination test, and the rate of positive infection is 19.5%.Serosurvey shows, there is MS and infect in domestic multiple area chicken groups.Prevention chicken Mycoplasma synoviae infects mainly suitable medicine input the most clinically, prevention primary disease is played a role, but for having occurred in that typical clinical symptoms, the effect of drug application is the most inconspicuous, the chicken Mycoplasma synoviae in chicken body can not be eradicated, it is easy to produce drug resistance, adds difficulty to the control of this disease.Prepare vaccine to carry out immunoprophylaxis being one of effective way preventing and treating primary disease with chicken Mycoplasma synoviae bacterial strain, the most domestic chicken Mycoplasma synoviae vaccine that there is no lists.
Summary of the invention
The technical problem to be solved is to provide a strain chicken Mycoplasma synoviae, and this vaccine strain has stronger virulence and the highest immunogenicity.
The chicken Mycoplasma synoviae YBF-MS1 (Mycoplasma synoviae YBF-MS1) of the present invention, it is preserved in the China typical culture collection center being positioned at Wuhan, Wuhan University, preservation date is on March 3rd, 2016, and preserving number is: CCTCC NO:M 2016080.
Above-mentioned chicken Mycoplasma synoviae vaccine strain can be used for preparing vaccine；
Described vaccine, preferably inactivated vaccine.
The chicken Mycoplasma synoviae YBF-MS1 strain of the present invention has stable biological characteristics, has strong virulence, and has good immunogenicity.Apply its oil adjuvant killed vaccine prepared safe and reliable, it is possible to producing the antibody of higher level, the duration is long, has intersecting protective, and after immunity, the sickness rate of chicken Mycoplasma synoviae significantly reduces, it is possible to effectively prevention chicken Mycoplasma synoviae is popular.The domestic chicken Mycoplasma synoviae vaccine that there is no lists, and prepares, with the chicken Mycoplasma synoviae YBF-MS1 strain of the present invention, the key that chicken Mycoplasma synoviae inactivated vaccine is this disease of prevention and control, also can overcome the disadvantages that gaps in market simultaneously.
Detailed description of the invention
According to example below, the present invention be may be better understood.But, as it will be easily appreciated by one skilled in the art that the content described by embodiment is merely to illustrate the present invention, and should be also without limitation on the present invention described in detail in claims.
Embodiment 1: the isolated and purified and qualification of chicken Mycoplasma synoviae YBF-MS1 strain.
1 chicken Mycoplasma synoviae production culture medium
1.1 improvement CM fluid mediums preparations take PPLO meat soup 21.0g, glucose 5g, deionized water add to 1000ml, 1% phenol red solution 1ml, after mentioned component mixed dissolution, 116 DEG C of autoclavings 30 minutes, after basal liquid cooling, aseptic addition inactivates horse serum 200ml, 25% yeast leachate 200ml, 800,000 units/ml penicillin solution 1.2ml, 1% nadide solution 30ml, 1%L-cysteine solution 30ml, adjust pH value to 7.6～7.8,2～8 DEG C of preservations with the sodium hydroxide solution of 1mol/L.
1.2 improvement CM solid mediums preparations take PPLO meat soup 21.0g, glucose 5g, agar powder 15.0g deionized water add to 1000ml, the dissolving of mentioned component mixing post-heating, quantitative separating, after 116 DEG C of sterilizings 30 minutes, put 2～8 DEG C of preservations.By 100ml solid medium heating for dissolving before using, when temperature drops to about 60 DEG C, add inactivation horse serum 20.0ml, 25% yeast leachate 20ml, 1% nadide solution 3.0ml, 1%L-cysteine solution 3.0ml, 80,000 units/ml penicillin 1.2ml.Adjust Medium's PH Value to 7.6～7.8,2～8 DEG C of preservations with the sodium hydroxide solution of 1mol/L.
2 chicken Mycoplasma synoviaes separate, identify
2.1 pathogenicbacteria separations:
In March, 2015, in chicken farm, Qingdao one, inject and the chicken group of chicken Mycoplasma synoviae vaccine there occurs typical infectiousness synovitis symptom.Swelling tarsal joint, vola and Os Draconis cyst content is taken from morbidity chicken, it is inoculated in improvement CM fluid medium, put 37 DEG C, 150r/min shaken cultivation 24～48 hours, the color of the fluid medium after to be seeded by scarlet become orange time results, pass on, take 2nd generation (F2 generation) bacterium solution and identify.
2.2 pathogen identification
2.2.1 morphologic observation chicken Mycoplasma synoviae separation strain (named: YBF-MS1 strain) is through Giemsa staining, and thalli morphology is polymorphic spherical bacterium.
2.2.2 biochemical characteristic chicken Mycoplasma synoviae separation strain YBF-MS1 strain energy glucose fermentation and maltose, produces acid not aerogenesis；Azymic lactose, it is impossible to decompose arginine；L-form is identified and is feminine gender；Can the erythrocyte of coagulation chicken.
2.2.3 cultural character chicken Mycoplasma synoviae separation strain YBF-MS1 strain well-grown in the improvement CM fluid medium containing nadide, cultivate 24～30 hours, growing in slight haziness, culture medium color is become orange (Medium's PH Value decline 0.7 and more than) by scarlet；Inoculation, containing on the improvement CM solid medium of nadide, puts 5%～10%CO2Under the conditions of, to cultivate 3～5 for 37 DEG C, visible circular, protuberance under low power lens, ganoid bacterium colony, colonies typical is in " fried egg shape ".This bacterium does not grows in the culture medium do not contain nadide.
2.2.4 purely inspection chicken Mycoplasma synoviae separation strain YBF-MS1 strain is tested by version " Chinese veterinary pharmacopoeia " annex in 2010, and result is purely.
2.2.5 specificity
2.2.5.1 test addition chicken Mycoplasma synoviae positive serum in the improvement CM fluid medium added with chicken Mycoplasma synoviae YBF-MS1 strain bacterium solution is resisted in metabolism, occurs in that specific metabolic inhibitory action, culture medium color do not change.
2.2.5.2 adding chicken Mycoplasma synoviae positive serum in the growth inhibition test improvement CM solid medium added with chicken Mycoplasma synoviae YBF-MS1 strain bacterium solution, occur in that specific metabolic inhibitory action, culture medium has no mycoplasma colony growth.
2.2.5 molecular biology identification chicken Mycoplasma synoviae specific primer carries out PCR amplification, and the strain of result YBF-MS1 can amplify the specific fragment of about 595bp.
Chicken Mycoplasma synoviae is separated YBF-MS1 strain and is shown by above every qualification result, all meets chicken Mycoplasma synoviae characteristic.
2.3 chicken Mycoplasma synoviae YBF-MS1 strain VlhA gene sequencing and analyses
2.3.1 design of primers is according to the chicken Mycoplasma synoviae VlhA gene included in GenBank, for conserved regions design primer after comparison, Dalian treasured biological engineering company limited synthesizes.This primer amplification clip size is 595bp, and primer sequence is as follows:
FvolA:CATTGCTCCTGCTGGTATAG
RvolA:TTGGAAGTCAAGTCCTGGTT
2.3.2 the preparation of DNA profiling takes YBF-MS1 strain culture 5.0ml, and 12000r/min is centrifuged 15min, abandons supernatant, precipitation 1.0ml distilled water is resuspended, and after 100 DEG C of water-bath 10min, 12000r/min is centrifuged 15min and collects supernatant, being pcr template ,-20 DEG C save backup.
2.3.3 PCR amplification and order-checking PCR amplification system are: 10 × PCR Buffer 2.5 μ l, dNTPs 1.0 μ l, primers F 0.5 μ l, primer R 0.5 μ l, template DNA 5.0 μ l, Taq enzyme 0.25 μ l, distilled water 15.25 μ l.Reaction condition is: 95 DEG C of denaturations 5min, 94 DEG C of degeneration 30s, 50 DEG C of annealing 30s, and 72 DEG C extend 1min, 35 circulations, and 72 DEG C extend 5min eventually.Take 10 μ l pcr amplification products, agarose gel with 1% is electroresis appraisal under 120V voltage, simultaneously using type strain as positive control, result occurs in that the specific band of the about 595bp identical with canonical reference strain WVU1853 strain, illustrates that this separation strain is chicken Mycoplasma synoviae.
2.3.4 check order and analyze according to the chicken Mycoplasma synoviae VlhA gene order included in GenBank, the pair of primers for conserved regions design, and expand, the VlhA genetic fragment of about 595bp size is amplified through order-checking；Sequence analysis shows that the homology of the isolated strains in the mycoplasma strains place different from other of this screening is only 95%.
Viable bacteria amount is about 10 by 2.4 virulence tests6CCU/ml chicken synovial capsule support body YBF-MS1 strain bacterium solution, footpad injection 49 age in days SPF chicken 10,0.2ml/ is only；Separately setting 10 to compare, footpad injection culture medium, 0.2ml/ is only.Raise routinely after counteracting toxic substances, observe 14, record test chicken incidence.
Counteracting toxic substances result shows, the clinical symptoms predominantly counteracting toxic substances pawl of morbidity chicken dare not land or walk lamely；Classical symptom is ball portion, toe joint swelling；There is tarsal joint swelling in indivedual morbidity chickens, and lassitude, feather are slightly disorderly；All there is not respiratory symptom in all counteracting toxic substances chickens.Morbidity chicken toe joint and ball portion cut open the visible granulation hyperplasia tissue of inspection, mycopus or cellulosic cheese thing, the visible canescence mycopus of inspection or cellulosic caseous substance are cutd open in tarsal joint swelling position, cut open the organs such as inspection all counteracting toxic substances chicken air bags, trachea, lungs and are showed no exception.The results are shown in Table 1.
Table 1: virulence test result
By concentration bacterium solution qualified for chicken Mycoplasma synoviae YBF-MS1 strain inactivation inspection, (before inactivation, viable bacteria amount is about 5 × 10 to 2.5 immunogenic mensuration9CCU/ml), add 5% tween 80 and prepare aqueous phase, according to 2:3 ratio and oil phase mixing and emulsifying, prepare inactivated vaccine.Respectively with each 10 of 0.1ml, 0.25ml, 0.5ml cervical region subcutaneous injection 21 age in days SPF chicken, separately set not immunized controls 10.Latter 28 days of immunity, each group test chicken is all about 10 by viable bacteria amount6The YBF-MS1 strain bacterium solution footpad injection counteracting toxic substances of CCU/ml, every 0.2ml.Test chicken incidence in observing 14.Result of the test shows, with 0.1ml immunity 21 age in days SPF chickens, the protective rate exempting from latter 28 days test chickens is 4/10；With 0.25ml and 0.5ml immunity 21 age in days SPF chickens, the protective rate exempting from latter 28 days test chickens is 8/10 and 9/10；Comparison chicken 8/10 falls ill.From table 2 result, the minimum immunoprotection dosage of chicken Mycoplasma synoviae YBF-MS1 strain is 0.25ml, the results are shown in Table 2.
Table 2: Study On Immunogenicity result
Embodiment 2:YBF-MS1 strain application in chicken Mycoplasma synoviae vaccine
1 strain seedling strain is chicken Mycoplasma synoviae YBF-MS1 strain, the thalline of this invention bacterial strain, colonial morphology, the biochemical special biological characteristics all meeting chicken Mycoplasma synoviae, has stronger virulence and good immunogenicity to Sensitivity age chicken.
Prepared by 2 production strains
Freeze-dried semen improvement CM fluid medium in chicken Mycoplasma synoviae YBF-MS1 strain basis is restored by 2.1 first order seed breedings, by 10%, seed liquor is inoculated in fluid medium respectively, put 37 DEG C, 150r/min shaken cultivation 24～30 hours (treating that culture medium color is become orange stopping cultivation by scarlet), results culture, through the most after the assay was approved, as first order seed.Less than-15 DEG C save backup.
2.2 secondary seeds and multistage seminal propagation take primary seed solution by 10% inoculation fluid medium, put 37 DEG C, 150r/min shaken cultivation 24～30 hours (treating that culture medium color is become orange stopping cultivation by scarlet) results cultures, after microscopy is qualified, as secondary seed, put 2～8 DEG C and save backup.The most progressively do seed amplification culture, until required grain weight.After microscopy is pure, as amplification culture seed liquor.
Prepared by 2.3 seedling bacterium solution
2.3.1 bacterium solution is cultivated and is cultivated by 10% addition chicken Mycoplasma synoviae YBF-MS1 strain seed liquor, cultivation temperature is 37 DEG C, 150r/min shaken cultivation 24～30 hours (treating that culture medium color is become orange stopping cultivation by scarlet) are gathered in the crops and are sampled, and carry out purely checking and count plate according to version " People's Republic of China's veterinary drug allusion quotation " annex in 2010.Every batch of preparation bacterium solution all should purely, and without varied bacteria growing, and viable count is all not less than 109CCU/ml。
2.3.2 bacterium solution concentration uses the method for continuous flow centrifugation that bacterium solution is made 5 times of concentrations, inactivates immediately after concentration.
2.3.3 in bacterium solution inactivation will concentrate bacterium solution aseptic importing inactivation bottle, according to concentrating bacterium solution quantity, metering adds formalin, make it be sufficiently mixed, final concentration of the 0.2% of formalin, 37 DEG C of shaking inactivations 12～16 hours (reach 37 DEG C with temperature in bottle and start timing), inactivated bacterial liquid through inactivation after the assay was approved, is put 2～8 DEG C of preservations and is used as antigen for vaccine.
Prepared by 2.4 vaccines
2.4.1 oil phase preparation takes injection white oil 95 parts, aluminium stearate 1 part, Si Ben-80 5 parts, and mix homogeneously is through 115 DEG C of sterilizings 40 minutes, standby after cooling.
2.4.2 aqueous phase preparation is by concentration bacterium solution 95 parts qualified for inactivation inspection, adds aseptic tween 80 5 parts, and fully mixing is aqueous phase.
2.4.3, in emulsifying takes oil phase 2 parts importing emulsion tank, start motor stirring at low speed, be slowly added into aqueous phase 1 part, 4000r/min emulsifying 30 minutes simultaneously.Sampling 10ml, 3000r/min are centrifuged 15 minutes, and the aqueous phase separated out at the bottom of pipe should be not more than 0.5ml.
2.4.4 subpackage quantitative separating, seals, 2～8 DEG C of preservations.
2.5 vaccine product inspections
2.5.1 character
2.5.1.1 outward appearance chicken Mycoplasma synoviae inactivated vaccine (YBF-MS1 strain) outward appearance is milky Emulsion.
2.5.1.2 dosage form water-in-oil type.Take a cleaning suction pipe, draw a small amount of vaccine and instill cold water surface, in addition to the 1st, equal indiffusion, stable dosage forms is described.
2.5.1.3, during stability draws vaccine 10ml addition centrifuge tube, 3000r/min is centrifuged 15 minutes, and the aqueous phase separated out at the bottom of pipe is not more than 0.5ml.
2.5.1.4 viscosity is tested by version " Chinese veterinary pharmacopoeia " annex in 2010, meets regulation.
2.5.2 loading quantity inspection is tested by version " Chinese veterinary pharmacopoeia " annex in 2010, meets regulation.
2.5.3 steriling test is tested by version " Chinese veterinary pharmacopoeia " annex in 2010, vaccine asepsis growth.
2.5.4 safety verification is with 22 age in days SPF chicken 10, every cervical region subcutaneous injection vaccine 1.0ml (2 plumage part), observes 14, and all test chickens are spiritual, searching for food and drinking water is showed no obvious abnormalities, and injection site and whole body all have no adverse reaction.
2.5.5 residual formaldehyde measure vaccine by 2010 version " Chinese veterinary pharmacopoeia " test, result shows, in vaccine, residual formaldehyde is 0.07, meets the regulation of veterinary biologics general rule.
2.5.6 efficacy test is with 21 age in days SPF chicken 20, is randomly divided into 2 groups, often group 10.Wherein 1 group of cervical region subcutaneous injection chicken Mycoplasma synoviae inactivated vaccine (YBF-MS1 strain), every 0.5ml；Another 1 group the most immune compares.All test chickens are taken a blood sample by latter 28 days of immunity, detect flat board agglutinating antibody；All test chickens are all about 10 by viable bacteria amount simultaneously6The chicken Mycoplasma synoviae YBF-MS1 strain bacterium solution footpad injection of CCU/ml carries out counteracting toxic substances, every 0.2ml.Result shows, latter 28 days of immunity, and immunity chicken 9/10 agglutinating antibody titer is not less than 1:16；Comparison chicken 10/10 agglutinating antibody titer is feminine gender；9/10 protection after immunity chicken counteracting toxic substances；9/10 morbidity after comparison chicken counteracting toxic substances.Vaccine potency test is qualified, illustrates that the inactivated vaccine prepared by chicken Mycoplasma synoviae YBF-MS1 strain has extraordinary immune protective efficiency.
Contrast experiment shows, carries out immunity with the chicken Mycoplasma synoviae vaccine sold in the market, then carries out challenge viral dosage with the YBF-MS1 strain of present invention screening；Result shows that the immune effect of the vaccine sold in the market is far below the effect of the inactivated vaccine that YBF-MS1 strain of the present invention is made.It is presumably due to YBF-MS1 pnca gene morph and cause the immune effect of current vaccine bad.
Claims (4)
1. a chicken Mycoplasma synoviae, it is characterised in that the deposit number of described chicken Mycoplasma synoviae
For CCTCC M 2016080.
2. chicken Mycoplasma synoviae as claimed in claim 1, it is characterised in that described chicken synovial fluid rami capsulares
The bacterial strain number of substance is YBF-MS1 strain.
3. the application in preparing vaccine of the chicken Mycoplasma synoviae described in claim 1.
Apply the most as claimed in claim 3, it is characterised in that described vaccine is inactivated vaccine.
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Cited By (5)
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| CN107384837A (en) * | 2017-09-02 | 2017-11-24 | 河南省农业科学院畜牧兽医研究所 | One plant of chicken synovia mycoplasma and its application |
| CN111537714A (en) * | 2020-06-01 | 2020-08-14 | 扬州大学 | Freeze-dried hemagglutination inhibition test antigen for detecting mycoplasma synoviae, antigen combination and preparation method |
| CN113755368A (en) * | 2021-08-17 | 2021-12-07 | 福建农林大学 | Fujian chicken bursa of synovium mycoplasma and culture medium thereof |
| CN115746109A (en) * | 2022-10-31 | 2023-03-07 | 扬州大学 | Chicken bursal mycoplasma synoviae protein RS00900 with nuclease function and application thereof |
| CN117568227A (en) * | 2023-11-29 | 2024-02-20 | 瑞普（保定）生物药业有限公司 | Mycoplasma synoviae vaccine strain, inactivated vaccine and preparation method thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107384837A (en) * | 2017-09-02 | 2017-11-24 | 河南省农业科学院畜牧兽医研究所 | One plant of chicken synovia mycoplasma and its application |
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| CN111537714A (en) * | 2020-06-01 | 2020-08-14 | 扬州大学 | Freeze-dried hemagglutination inhibition test antigen for detecting mycoplasma synoviae, antigen combination and preparation method |
| CN111537714B (en) * | 2020-06-01 | 2023-04-18 | 扬州大学 | Freeze-dried hemagglutination inhibition test antigen for detecting chicken mycoplasma synoviae, antigen combination and preparation method |
| CN113755368A (en) * | 2021-08-17 | 2021-12-07 | 福建农林大学 | Fujian chicken bursa of synovium mycoplasma and culture medium thereof |
| CN115746109A (en) * | 2022-10-31 | 2023-03-07 | 扬州大学 | Chicken bursal mycoplasma synoviae protein RS00900 with nuclease function and application thereof |
| CN115746109B (en) * | 2022-10-31 | 2024-02-27 | 扬州大学 | Chicken bursa mycoplasma synoviae protein RS00900 with nuclease function and application thereof |
| CN117568227A (en) * | 2023-11-29 | 2024-02-20 | 瑞普（保定）生物药业有限公司 | Mycoplasma synoviae vaccine strain, inactivated vaccine and preparation method thereof |
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